Mechanistic inhibition studies of SARS-CoV replication and entry

Abstract

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Severe acute respiratory syndrome (SARS) is an infectious and highly contagious disease that is caused by SARS-associated coronavirus, (SARS-CoV) and for which there are currently no approved treatments. The non-structural protein 13 (nsp13) of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) is a helicase that separates double-stranded RNA (dsRNA) or DNA (dsDNA) with a 5'->3' polarity, using the energy of nucleotide hydrolysis. We determined the minimal mechanism of helicase function by nsp13. We showed a clear unwinding lag with increasing length of the double-stranded region of the nucleic acid, suggesting the presence of intermediates in the unwinding process. Our data provide experimental evidence that nsp13 and nsp12 can function in a concerted manner to improve the efficiency of viral replication and enhance our understanding of nsp13 function during SARS-CoV RNA synthesis. We also report the discovery of a potent inhibitor of SARS-CoV that blocks replication by inhibiting the unwinding activity of the SARS-CoV helicase (nsp13). Using the identified nsp13 inhibitor, we investigate the role of helicase in SARS-CoV life cycle and our results show that nsp13 interferes with positive strand synthesis with no effect on the negative strand synthesis. Finally, we report the discovery of potent inhibitors of SARS-CoV replication that block entry of SARS-CoV by three different mechanisms. We propose that these compounds will be a valuable tool for further understanding SARS-CoV life cycle, which could be a model for other nidoviruses, and also candidates for further development as a SARS antiviral.